[MUSIC] Now we're going to make a huge jump in time and go to 1999. Probably all of you were born by 1999, or at least at the time of this recording. Now we are in the era of genome sequences. And people have sequenced genomes of different types of Streptococcus pneumonia. And this sequence data is actually quite fascinating. We've heard about Type 2 and Type 3 pneumococcus. Type 2 is of pneumococcus from which the R strain was derived. And Type 3 is of thermococcus from which the DNA was purified. Now you may consider that Avery and his colleagues were extremely lucky. But in fact, yes they were lucky. But they also knew not to choose the most complicated system but to choose the most simple system to try to provide the transforming principle. But they were lucky. Here are the genes of what is called today a pathogenicity island. It's a bunch of genes together that will go from one bacterium to another bacterium, move together as a package and confer virulence. And so each of the types of pneumococcus has a different pathogenicity island. And now you can see that Type 1 has many genes. It goes from gene A to K to 1 through 4, to LEA, okay? LEA is outside the locals, so it's all of these genes are necessary or at least are present in the Type 1 isolates. The Type 2 isolate is slightly different and we're not going to worry about the symbols, each arrow is a gene, gene A is one gene because there is one arrow. Type 2 has a different structure, but it has A, B, C, D, E, T, because T was discovered later to be between E and F, G, H, Y, J, K, P, L, M, N, O. All of these genes are in this pathogenicity island. And Griffith has actually observed early on, that the Type 2, for reasons that he didn't understand, and we still not completely understand today, the Type 2 are easier to transform than the Type 3. So because of that and because Avery's group has discovered, made the same observation. They decided that they were going to use an R derived from a Type 2. Just to make things a little bit easier for them and they picked this strain that you've seen when you read the paper, R36A. Now, R36A was sequenced and R36A was predicted to be a deletion because it doesn't revert, too smooth, too virulent. And in fact, it's a deletion of 7 kilobase. 7,000 base pairs. The extent of this deletion is indicated by this dark bar here. So in fact, this strain removes a number of genes, but not all the genes and that will come back to not all the genes. Type 3, the Type 3 capsule is the simplest capsule and Type 3 virulence pathogenicity island is the simplest of all these islands. And in fact, it only contains three genes that were called a, b and c. What are these genes doing? Well, these genes are helping to make the capsules. So we use a molecule of glucose and we activate this molecule of glucose. This is gene C is making an activated glucose. For those of you who like chemistry, this activated glucose is uridine diphosphoglucose UDP Glu, that's gene C. Then comes the second gene, is a gene A. Gene A will convert this UDP Glu into UDP-Gla, for glucuronic acid. It's a very simple chemical reaction catalyzed by this enzyme A. And then enzyme B will make a polymer. That is, it will take many glucuronic acid and glucose and put them together to make a chain. The same way you make nylon and you make plastic ware. So if you want to make a Type 3, you need three genes. Gene C, gene A, gene B, okay? Now in this particular case, Streptococcus pneumonia has another gene which is equivalent to C and this gene for some historical reason is called galU. GalU does the same job as C. C is just redundant. So you don't need to have the C gene to make a Type 3 capsule. Because the activity of the C gene is provided by the normal chromosome of Streptococcus, virulent or non-virulent. So you don't really need C. Now, this gene is required to make the capsule, gene A. But, deletion R36A removes a portion of this Type 2 here and leaves this gene, which is called CPS2K intact. And K has the same activity as gene A from Type 3. So it's basically you get one screwdriver from one box and then you don't have this tool box with you but you have another tool box with another screwdriver provided that the size of the screwdriver is more or less okay. You can use the other box. This is sometimes called gene redundancy. So basically, the only thing that is required to make a Type 3 capsule is this gene. This gene is about one kilobase long. This is about the size of the DNA fragments that Avery and his colleagues were able to. They don't need more. They were lucky, because they use a simple system and they were lucky because the deletion they used helped them to have an even smaller size of fragment of DNA than what was required. It is luck, but if they didn't have anything that worked, they would have tried something else. They were adamant in getting transformation to work with purified DNA and it worked. In spite of the difficulties, in spite of the time, it worked. This 50 years later, when people got this sequence information, they realized that Avery was lucky. But it was some kind of a relief that the system was so flexible that if they had to transform with a large piece of DNA at 20 kilobase long, DNA's much harder. It could have been done, but it certainly would have taken several more years. They were lucky but they knew how to pick their luck.
